Compositions and methods comprising lysin plycp025 and derivatives thereof

ABSTRACT

Provided are compositions and methods for use in selectively killing one or more of C. perfringens, C. sordelli and C. histolyticum. The compositions include lysin PlyCP025, as well as catalytically active fragments thereof, and variants thereof that retain killing activity. Methods for reducing one or more of C. perfringens, C. sordelli or C. histolyticum bacteria are provide and involve contacting such bacteria with a composition that contains PlyCP025 or an enzymatically active fragment or variant thereof, which can be provided as recombinant polypeptides. The composition and methods are useful for human and veterinary purposes. Diagnostic approaches are also included by contacting a sample obtained or derived from an animal, with a recombinant polypeptide, and detecting binding of the polypeptide to bacteria in the sample if said bacteria that are bound to the polypeptide are present in the sample. The polypeptide may thus be detectably labeled to produce a detectable signal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/598,039, filed on Dec. 13, 2017, the disclosure of which is herebyincorporated by reference.

FIELD

The present disclosure relates generally to methods, compositions andarticles of manufacture useful for the prophylactic and therapeuticamelioration and treatment of Gram-positive bacteria, including but notnecessarily limited to Clostridium perfringens bacterial strains. Thedisclosure relates to compositions and articles of manufactureincorporating isolated and engineered C. perfringens bacteriophagelysins including variants and truncations thereof, and to methodsutilizing the lysin polypeptides and compositions.

BACKGROUND

Clostridium peifringens, a Gram-positive, rod-shaped, anaerobic.spore-forming bacterium. C. perfringens may be found as a component ofdecaying vegetation and the intestinal tract of humans and othervertebrates, insects, and in the soil. C. perfringens can cause severeinfections of animals and humans, and is known to be the cause of foodpoisoning, gas gangrene, necrotic enteritis, and gastrointestinalinfections not associated with food. Generally, C. perfringens is apathogen that affects both animals and humans. Although C. perfringenshas been known to infect all animals it is a particularly prevalentpathogen in the poultry industry.

C. perfringens causes necrotic enteritis in chickens. Clinical illnessis usually short lived but the mortality in poultry flocks can be highwith obvious financial and animal health implications. Sometimes theonly sign of necrotic enteritis is a sudden increase in mortality.However, necrotic enteritis may cause birds to appear depressed, haveruffled feathers, and dark diarrhea. The disease may persist in a flockfor about 5-10 days, with up to 50% mortality.

Antimicrobial drugs delivered at prophylactic doses in water or feed maycontrol necrotic enteritis. However, there is increasing publicopposition to the use of antibiotics in animal husbandry. But withouttraditional antibiotics to prevent necrotic enteritis, this diseasecould potentially become a greater problem for the poultry industry.Thus, there is a need to identify new ways to prevent or treatinfections by C. perfringens.

SUMMARY OF THE DISCLOSURE

The present invention provides compositions and methods for use inselectively killing certain types of bacteria. Non-limiting embodimentsare implemented using a lysin referred to herein as PlyCP025, includingcatalytically active fragments thereof, and variants thereof that retainkilling activity. In this regard, the lysin comprises a lytic enzymesegment, and thus contains a lytic polypeptide sequence that is suitablefor treating and/or preventing C. perfringens infections, as well asinfection by certain other bacteria, including but not necessarilylimited to C. sordelli and C. histolyticum. However, the samepolypeptides exhibits less, or no detectable activity against C.septicum, C. bifermentans and C. difficile and certain other bacteria.Thus, the compositions and methods are useful for selectively killingbacteria in a targeted manner.

In non-limiting embodiments, the disclosure includes a method forreducing one or more of C. perfringens, C. sordelli or C. histolyticumbacteria. The method generally comprises contacting the bacteria with acomposition comprising a lytic enzyme described herein. In embodiments,the lytic enzyme comprises the amino acid sequence of SEQ ID NO: 2 or anamino acid sequence with a contiguous segment having at least 95%identity to the amino acid sequence of SEQ ID NO:2 such that one or moreof the one or more of the C. perfringens, C. sordelli or C. histolyticumbacteria are killed. In embodiments, the lytic enzyme consists of thesequence of SEQ ID NO:2 or an amino acid sequence with a contiguoussegment having at least 95% identity to the amino acid sequence of SEQID NO:2. In certain aspects, any one or a combination of C. perfringens,C. sordelli or C. histolyticum bacteria are killed. In embodiments, atleast C. perfringens bacteria are killed. In embodiments, any one or anycombination of the bacteria are antibiotic resistant and are killed. Inembodiments, the bacteria are killed are in or on a human, or in or on anon-human animal selected from avian animals and non-human mammals. Inembodiments, the bacteria are in the individual in a population ofbacteria that further comprise commensal gut bacterium, but thecommensal gut bacteria are not killed by the lytic enzyme. In certainapproaches, the commensal bacteria are selected from C. septicum, C.novyi, E. faecalis, E. faecium, L. rhamnosous, and combinations thereof.

In another embodiment, the disclosure provides a pharmaceuticalformulation, which may be provided as a veterinary formulation, forkilling C. perfringens, C. sordelli or C. histolyticum, or a combinationthereof. The formulation(s) comprise a lytic enzyme described herein,and at least one pharmaceutically acceptable carrier or excipient.

In another embodiment, the disclosure provides a method of making arecombinant polypeptide capable of killing C. perfringens, C. sordelliand C. histolyticum as described herein. This approach comprisesexpressing the recombinant polypeptide in a population of cellscomprising an expression vector that encodes and expresses therecombinant polypeptide, allowing expression of the recombinantpolypeptide, and separating the recombinant polypeptide from thepopulation of cells. The expression vectors, and cells comprising theexpression vectors are also included.

In certain aspects the disclosure comprises one or more bacteria, suchas a population of bacteria, that are in physical association with arecombinant polypeptide described herein. In embodiments, such bacteriaare present on a mammal, or on an avian animal.

Diagnostic approaches are also included. Diagnostic approaches comprisecontacting a sample obtained or derived from an animal, such as a mammalor an avian animal, with a polypeptide described herein, and detectingbinding of the polypeptide to bacteria in the sample if said bacteriathat are bound to the polypeptide are present in the sample. Innon-limiting examples, the polypeptide is detectably labeled, and asignal from the detectable label is detected if bacteria that are boundto the polypeptide are present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The colony-forming unit (CFU) reduction of C. perfringensATCC13124 by PlyCP025cat.

FIG. 2A. The effect of pH on the lytic activity of PlyCP025cat. Thelytic activity was determined by OD600 reduction over 60 minutes. Foreach sample, 30 μg of lysin or equal volume of phosphate buffer (PB) wasadded to C. perfringens ATCC13124 resuspension (20 mM PB, pH 6, 7, and 8respectively).

FIG. 2B. The effect of NaCl on the lytic activity of PlyCP025cat. Thelytic activity determined by OD600 reduction over 60 minutes. For eachsample, 30 μg of lysin or equal volume of PB is added to C. perfringensATCC13124 resuspension (20 mM PB, pH 8, with the addition of 20 mM NaCl,100 mM NaCl, 200 mM NaCl, 400 mM NaCl and 500 mM NaCl respectively).

FIG. 3. Host specificity of PlyCP025cat. The lytic activity ofPlyCP025cat was assessed in a variety of bacteria species, includingother clostridia and other common gut flora bacteria. The effect ofPlyCP025cat is measured by calculating the OD600 ratio between 60 minand the starting value

FIG. 4. Graph showing comparison of the activity of PlyCP025 andPlyCP025cat.

DETAILED DESCRIPTION

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure pertains.

Unless specified to the contrary, it is intended that every maximumnumerical limitation given throughout this description includes everylower numerical limitation, as if such lower numerical limitations wereexpressly written herein. Every minimum numerical limitation giventhroughout this specification will include every higher numericallimitation, as if such higher numerical limitations were expresslywritten herein. Every numerical range given throughout thisspecification will include every narrower numerical range that fallswithin such broader numerical range, as if such narrower numericalranges were all expressly written herein.

The present invention provides compositions and methods related to alysin referred to herein as PlyCP025. The lysin comprises a lytic enzymeand thus contains a lytic polypeptide sequence that is suitable fortreating and/or preventing C. perfringens infections, as well asinfection by certain other bacteria. In this regard, it is believed thatPlyCP025 and a catalytic fragment of it exhibit effective lytic activityagainst C. perfringens strains, as well as against C. sordelli and C.histolyticum. However, the same polypeptides display little to noactivity against C. septicum, C. bifermentans and C. difficile. Thus, inembodiments, a composition of this disclosure exhibits greater lyticactivity against C. perfringens relative to any suitable referencevalue, including but not necessarily limited to lytic activity againstone or combination of C. septicum, C. bifermentans and C. difficile.Lytic activity can be measured using any suitable approach. Inembodiments, lytic activity is measured via a change in CFU.

As used herein a “lytic enzyme” and “lytic polypeptide sequence” is abacterial cell wall lytic enzyme that kills and/or is capable of killingone or more bacteria under suitable conditions and during a relevanttime period. In particular embodiments a composition of the disclosurecomprises a contiguous segment or variant of the polypeptide encoded bya PlyCP025 gene, the sequence of which is provided under NCBI database(WP_025647622.1), from which the nucleotide sequence is incorporatedherein by reference as it exists on the filing of this date of thisapplication or patent. The amino acid sequence of this polypeptide is:

(SEQ ID NO: 1)

EVENKPESVPSNTETSNTYFRVVVGSY KDRENAVKKQEELKAKGEDSFLLAYKE,wherein the catalytic domain is shown in bold and italics. A polypeptidethat has the sequence of SEQ ID NO:1 is referred to herein from time totime as PlyCP025.

In embodiments, the disclosure comprises a lytic polypeptide derivedfrom PlyCP025, and thus may comprise a contiguous segment or variant ofthe catalytic domain of PlyCP025. The catalytic domain of PlyCP025 whichis annotated above in bold and italics has the sequence:

(SEQ ID NO: 2) MSKIFGLDAGHCTSGADTGAQGNGYKEQDLTRQVVTYLSEYLEKEGHTTKYCHCNSASTVNESLRYRVNKANSIGVDYFVSIHLNAGGGVGTETYICARGGEAERVAKRVNSKLVQYGYRDRGVKVGNLYVIKNTNAPAILVEICFIDSSSDVAKFNAKAIAKAIAEGLLDKTIG,which is shown in bold an italics in the first 175 amino acids of SEQ IDNO:1. A polypeptide that is the catalytic domain of PlyCP025 is referredto herein from time to time as PlyCP025cat.

In embodiments, the present disclosure comprises polypeptides thatcomprise or consist of the sequence given for PlyCP025 or PlyCP025cat,or variants thereof. In embodiments, the polypeptide is capable ofbinding specifically to and/or lysing cells of C. perfringens strains,and may also be capable of lysing C. sordelli and C. histolyticum. Thus,the disclosure provides lytic polypeptides that are capable of killingsuch bacteria, for instance by having at least some cell wall lyticactivity against the host bacteria. In certain embodiments thepolypeptides may be extended to include, for example, amino acidsC-terminal to amino acid 175 in SEQ ID NO:1. In embodiments, between1-53 amino acids C-terminal to amino acid 175 in SEQ ID NO:1 can beincluded.

In certain implementations the disclosure includes polypeptides andmethods of using them, wherein the polypeptides comprise a functionallyactive lytic enzyme that shares a particular percent amino acid sequenceidentity with SEQ ID NO:1 and/or SEQ ID NO:2, and can kill for C.perfringens bacteria, and other susceptible bacteria as describedherein. Percent amino acid sequence identity with respect to the phageassociated lytic enzyme sequences identified is defined herein as thepercentage of amino acid residues in a polypeptide sequence of thisdisclosure that are identical with the amino acid residues in a phageassociated lytic enzyme sequence, after aligning the sequences in thesame reading frame and introducing gaps, if necessary, to achieve themaximum percent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. In embodiments,polypeptides of this disclosure have at least 60, 65, 70, 75, 80, 85,90, 95, 97, 98, 99 or 99.5% amino acid sequence, inclusive, andincluding all numbers there between to the first decimal point, identitywith SEQ ID NO:1 or SEQ ID NO:2. The disclosure further includespolypeptides comprising or consisting of SEQ ID NO:1 and SEQ ID NO:2 andpolypeptides having the stated amino acid sequence identity, wherein oneor more amino acid residues are added, or deleted, wherein such aminoacid insertions or deletions may be within the polypeptide, or at the Nor C terminus of the sequences.

Biologically active portions of polypeptides of this disclosure can alsocomprise mutations, provided such mutant proteins maintainapproximately, at least as much, or exhibit more lytic activity than apolypeptide comprising or consisting of SEQ ID NO:1 or SEQ ID NO:2. Suchmutants or variants thereof may have a predicted function and/or can betested for function or the capability to kill bacteria. Thus, changescan be made to the sequence of SEQ ID NO:1 and/or SEQ ID NO:2, forexample, and mutants or variants having a change in sequence can betested using the assays and methods described and exemplified herein,and/or as will be otherwise apparent to those skilled in the art. Thus,when given the benefit of the present disclosure, a skilled artisan canpredict one or more amino acids suitable for substitution or replacementand/or one or more amino acids which are not suitable for substitutionor replacement, including reasonable conservative or non-conservativesubstitutions. Certain substitutions include but are not limited to: Lysfor Arg and vice versa such that a positive charge may be maintained;Glu for Asp and vice versa such that a negative charge may bemaintained; Ser for Thr such that a free hydroxide can be maintained;and Gln for Asn such that a free amine can be maintained. Exemplaryconservative amino acid substitutions include any of: glutamine (Q) forglutamic acid (E) and vice versa; leucine (L) for valine (V) and viceversa; serine (S) for threonine (T) and vice versa; isoleucine (I) forvaline (V) and vice versa; lysine (K) for glutamine (Q) and vice versa;isoleucine (I) for methionine (M) and vice versa; serine (S) forasparagine (N) and vice versa; leucine (L) for methionine (M) and viceversa; lysine (K) for glutamic acid (E) and vice versa; alanine (A) forserine (S) and vice versa; tyrosine (Y) for phenylalanine (F) and viceversa; glutamic acid (E) for aspartic acid (D) and vice versa; leucine(L) for isoleucine (I) and vice versa; lysine (K) for arginine (R) andvice versa. Amino acid substitutions are typically of single residues,or can be of one or more, one or a few, one, two, three, four, five, sixor seven residues; insertions usually will be on the order of about from1 to 10 amino acid residues; and deletions will range about from 1 to 30residues. Deletions or insertions may be in single form, but typicallyare made in adjacent pairs, i.e., a deletion of 2 residues or insertionof 2 residues.

When used according to methods of this disclosure, PlyCP025 and variantsthereof (including but not limited to PlyCP025cat) may be used asexogenous agents that are uncoupled from a requirement for intracellularexpression, and are accordingly suitable for use in pharmaceuticalformulations that are described more fully below. Accordingly, incertain aspects, the disclosure provides a single bacterium, andpopulations of bacteria that are in physical association withpolypeptides of this disclosure. In certain embodiments, the disclosurecomprises a population of C. perfringens, and optionally C. sordelliiand B. subtilis and combinations thereof, wherein the bacterial cellscomprise a polypeptide of this disclosure in physical association with acomponent of peptidoglycan present in the bacteria. In embodiments, thepeptidoglycan may comprise N-deacylated glucosamine (N-deacylated NAG),N-acetylmuramic acid (NAM), N-deacylated NAM, or any combinationsthereof. In one embodiment, the peptidoglycan structure is comprised ofalternating NAM and NAG residues, which may be N-deacetylated, and wherea tripeptide, tetrapeptide, or pentapeptide bound to the NAM residues,is crosslinked between the third amino acid of one strand to the fourthamino acid, typically a D-alanine, of a tripeptide, tetrapeptide,pentapeptide bound to a NAM residue on a neighboring strand. In apreferred embodiment, the peptidoglycan structure is comprised ofalternating NAM and NAG residues, where the majority of NAG residues areN-deacetylated, and where a tripeptide, tetrapeptide, or pentapeptidebound to the NAM residues, is crosslinked between the third amino acidof one strand to the third amino acid of a tripeptide, tetrapeptide,pentapeptide bound to a NAM residue on a neighboring strand. Thus thedisclosure encompasses C. perfringens, and optionally C. sordellii andB. subtilis and combinations thereof, wherein a polypeptide of thisdisclosure has been introduced into a peptidoglycan layer of thebacteria exogenously, i.e., without being first expressed within thebacteria. The physical association between the polypeptide andpeptidoglycan component can be non-covalent, and can comprise, forexample, the polypeptide being adjacent to its peptidoglycan substratesuch that it can perform enzymatic cleavage of the substrate, and mayinclude cleavage intermediates, such as complexes formed between thepolypeptide and the substrate during cleavage.

The polypeptide may comprise a binding portion at the carboxyl terminalside and may be characterized by an enzyme activity capable of cleavingcell wall peptidoglycan (such as an amidase activity able to act onamide bonds in the peptidoglycan) at the amino terminal side. In anembodiment, a lysin of this disclosure comprises anN-acetylmuramoyl-L-alanine amidase.

The disclosure includes fusion proteins which comprise all, or abiologically active part, of a polypeptide according to the presentdisclosure linked to a heterologous polypeptide. Fusion proteins includechimeric proteins are produced, for example, by chemically combining twoor more distinct proteins, or as a continuous polypeptide made byprotein translation. These proteins can act independently on the same ordifferent molecules, and hence have a potential to treat two or moredifferent bacterial infections at the same time. These proteins may beused to treat a bacterial infection by cleaving the cell wall in morethan one location, thus potentially providing more rapid or effective(or synergistic) killing from a single lysin molecule. A fusion proteincan combine a lysin polypeptide with a protein or polypeptide of havinga different capability, or providing an additional capability or addedcharacter to the lysin polypeptide. Chimeric and fusion proteins andpolypeptides of the disclosure can be produced by standard recombinantDNA techniques.

Any of the polypeptides of this disclosure can be produced using anysuitable approach, examples of which are known in the art. In certainembodiments, the polypeptides can be isolated and/or separated from abacteriophage. In embodiments, the polypeptides are produced using anysuitable expression vector, such expression vectors also beingencompassed by the disclosure. Accordingly, the polypeptides may beproduced by using the isolated gene encoding the lytic enzyme from thephage genome, and/or cloned versions of the gene, or any polynucleotidesegment or derivative of it that encodes the polypeptides of thisdisclosure. The expression vector for producing a lysin polypeptide orenzyme of the invention may be suitable for expression in any suitablebacteria, including but not limited to E. coli, Bacillus, or any numberof other suitable bacteria. The vector and/or RNA encoding it may alsobe used in a cell free expression system. Accordingly, allpolynucleotides encoding polypeptides of this disclosure are encompassedwithin the invention, as are polynucleotides that hybridize, understringent conditions, with complementary sequences of the DNAsequence(s). Further variants of these sequences are contemplated foruse in production of lysin enzymes according to the disclosure,including natural variants that may be obtained. Thus, a large varietyof isolated nucleic acid sequences or cDNA sequences that encode phageassociated lysing enzymes and partial sequences that hybridize with suchgene sequences are useful for recombinant production of the lysinenzyme(s) or polypeptide(s) of the disclosure. In certain approaches asignal sequence can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell.

The present disclosure also provides pharmaceutical compositionscomprising the present polypeptides. Such compositions may be usedprophylactically or therapeutically. The pharmaceutical compositionscomprise one or more lytic polypeptide(s), and optionally includenatural, truncated, chimeric or shuffled lytic enzymes, optionallycombined with other components such as another polypeptide, apolynucleotide, holin protein(s), one or more antibiotics and/or as apharmaceutically acceptable carrier, excipients, carriers or vehicles.

The present disclosure provides therapeutic compositions orpharmaceutical compositions of the lysins for use in the killing,alleviation, decolonization, prophylaxis or treatment of theGram-positive bacteria described herein, including bacterial infectionsor related conditions. The present disclosure provides therapeuticcompositions or pharmaceutical compositions of the lysins of theinvention for use in treating, reducing or controlling contaminationand/or infections by Gram-positive bacteria, particularly including C.perfringens. Compositions are thereby contemplated and provided fortherapeutic applications and local or systemic administration.Additionally, different lytic polypeptide(s) genetically coded for bydifferent phage for treatment of the same bacteria may be used. Theselytic enzymes may also be any combination of naturally occurring lyticenzymes, truncated lytic polypeptide(s), variant lytic polypeptide(s),and chimeric and shuffled lytic enzymes. The lyticenzyme(s)/polypeptide(s) in a therapeutic or pharmaceutical compositionfor Gram-positive bacteria may be used alone or in combination withantibiotics or, if there are other invasive bacterial organisms to betreated, in combination with other phage associated lytic enzymesspecific for other bacteria being targeted. Various antibiotics may beoptionally included in the therapeutic composition with the enzyme(s) orpolypeptide(s). The pharmaceutical composition can also include a one ormore of a pharmaceutically acceptable carrier or diluent. More than onelytic enzyme or polypeptide may be included in the therapeuticcomposition. Additionally, compositions of this disclosure may furthercomprise other enzymes and/or mucolytic peptides, such as lysostaphin.

Compositions of this disclosure may further include at least oneadditional agent which can also potentiate the bactericidal activity ofthe composition. Antimicrobials act largely by interfering with thestructure or function of a bacterial cell by inhibition of cell wallsynthesis, inhibition of cell-membrane function and/or inhibition ofmetabolic functions, including protein and DNA synthesis. Antibioticscan be subgrouped broadly into those affecting cell wall peptidoglycanbiosynthesis and those affecting DNA or protein synthesis inGram-positive bacteria. Cell wall synthesis inhibitors, includingpenicillin and antibiotics like it, disrupt the rigid outer cell wall sothat the relatively unsupported cell swells and eventually ruptures.Antibiotics affecting cell wall peptidoglycan biosynthesis includeglycopeptides, which inhibit peptidoglycan synthesis by preventing theincorporation of N-acetylmuramic acid (NAM) and N-acetylglucosamine(NAG) peptide subunits into the peptidoglycan matrix. Availableglycopeptides include vancomycin and teicoplanin. Penicillins act byinhibiting the formation of peptidoglycan cross-links. The functionalgroup of penicillins, the β-lactam moiety, binds and inhibitsD,D-transpeptidase that links the peptidoglycan molecules in bacteria.Hydrolytic enzymes continue to break down the cell wall, causingcytolysis or death due to osmotic pressure. Common penicillins includeoxacillin, ampicillin and cloxacillin. Polypeptides interfere with thedephosphorylation of the C55-isoprenyl pyrophosphate, a molecule thatcarries peptidoglycan building-blocks outside of the plasma membrane. Acell wall-impacting polypeptide is bacitracin.

If the compositions include an antibiotic any suitable antibiotic can beadded, such as erythromycin, clarithromycin, azithromycin,roxithromycin, other members of the macrolide family, penicillins,cephalosporins, and any combinations thereof in amounts which areeffective to enhance (including but not limited to synergisticenhancement) the therapeutic effect of the lytic enzyme. Virtually anyother antibiotic may be used with the lytic enzymes of this disclosure.Therapeutic or pharmaceutical compositions may comprise lyticpolypeptide(s) combined with a variety of carriers to treat theillnesses caused by the susceptible bacteria. The carrier suitablycontains minor amounts of additives such as substances that enhanceisotonicity and chemical stability. Such materials are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, succinate, acetic acid, and otherorganic acids or their salts; antioxidants such as ascorbic acid; lowmolecular weight (less than about ten residues) polypeptides, e.g.,polyarginine or tripeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;glycine; amino acids such as glutamic acid, aspartic acid, histidine, orarginine; monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, glucose, mannose, trehalose, ordextrins; chelating agents such as ethylenediaminetetracetic aciddisodium salt (EDTA); sugar alcohols such as mannitol or sorbitol;counter-ions such as sodium; non-ionic surfactants such as polysorbates,poloxamers, or polyethylene glycol (PEG); and/or neutral salts, e.g.,NaCl, KCl, MgCl₂, CaCl₂), and others. Glycerin or glycerol(1,2,3-propanetriol) is commercially available for pharmaceutical use.It may be diluted in sterile water for injection, or sodium chlorideinjection, or other pharmaceutically acceptable aqueous injection fluid.The polypeptides may be provided in an environment having a controlledpH, which allows for activity of the lytic enzyme/polypeptide(s). Forexample if a human or a non-human animal has been exposed to anotherhuman or non-human animal with mucosal infection, the lyticenzyme/polypeptide(s) will reside in the mucosal lining and preventand/or inhibit colonization of the infecting bacteria. Prior to, or atthe time the altered lytic enzyme is put in the carrier system or oraldelivery mode, the polypeptide may be provided in a stabilizing bufferenvironment for maintaining a pH range between about 4.0 and about 9.0,more preferably between about 5.5 and about 8.5.

A stabilizing buffer may allow for the optimum activity of thepolypeptide(s). The buffer may contain a reducing reagent, such asdithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as EDTA, or it may also contain a phosphate orcitrate-phosphate buffer, or any other buffer. A mild surfactant can beincluded in a therapeutic or pharmaceutical composition in an amounteffective to potentiate the therapeutic effect of the lyticenzyme/polypeptide(s) may be used in a composition. Suitable mildsurfactants include esters of polyoxyethylenesorbitan and fatty acids(Tween series), octylphenoxypolyethoxy ethanol (Triton-X series),n-Octyl-β-D-glucopyranoside, n-Octyl-β-D-thioglucopyranoside,n-Decyl-β-D-glucopyranoside, n-Dodecyl-β-D-glucopyranoside, andbiologically occurring surfactants, e.g., fatty acids, glycerides,monoglycerides, deoxycholate and esters of deoxycholate.

Preservatives may also be used in this invention and may, for example,comprise about 0.05% to 0.5% by weight of the total composition. The useof preservatives assures that if the product is microbiallycontaminated, the formulation will prevent or diminish microorganismgrowth. Some preservatives useful in this invention includemethylparaben, propylparaben, butylparaben, chloroxylenol, sodiumbenzoate, DMDM Hydantoin, 3-Iodo-2-Propylbutyl carbamate, potassiumsorbate, chlorhexidinedigluconate, or a combination thereof.

In embodiments a therapeutically effective amount of a lytic enzyme isused. Therapeutically effective amount means that amount of apolypeptide of this disclosure that will elicit the biological ormedical response of a subject that is being sought by a health careprovider, regardless of whether for human or veterinarian purposes. Inparticular, with regard to Gram-positive bacterial infections and growthof Gram-positive bacteria, the term “effective amount” is intended toinclude an effective amount of a polypeptide of this disclosure thatwill bring about a biologically meaningful decrease in the amount of orextent of infection of Gram-positive bacteria, including having abactericidal and/or bacteriostatic effect. The phrase “therapeuticallyeffective amount” is used herein to mean an amount sufficient toprevent, and preferably reduce by at least about 30 percent, morepreferably by at least 50 percent, most preferably by at least 90percent, a clinically significant change in the growth or amount ofinfectious bacteria, or other feature of pathology such as for example,elevated fever or white cell count as may attend its presence andactivity. Such changes can be compared to changes in any suitablereference, such as a value determined by exposure of a similar amount tobacteria. Suitable controls and control values to determine, forexample, relative killing activity, will be apparent to those skilled inthe art given the benefit of the present disclosure.

Effective amounts of polypeptides of this disclosure will depend in parton whether the lytic enzyme/polypeptide(s) will be used therapeuticallyor prophylactically, the duration of exposure of the recipient to theinfectious bacteria, the size and weight of the individual, etc. Theduration for use of the composition containing the enzyme/polypeptide(s)also depends on whether the use is for prophylactic purposes, whereinthe use may be hourly, daily or weekly, for a short time period, orwhether the use will be for therapeutic purposes wherein a moreintensive regimen of the use of the composition may be needed, such thatusage may last for hours, days or weeks, and/or on a daily basis, or attimed intervals during the day.

For any lytic enzyme disclosed herein, the therapeutically effectivedose can be estimated initially either in cell culture assays or inanimal models, usually mice, rabbits, dogs, or pigs. The animal model isalso used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans or non-human animals, suchas for veterinary purposes. The exact dosage can be selected by theindividual health care provider in view of the individual to be treated.In certain embodiments, the effective dosage rates or amounts of thepolypeptide(s) to be administered, and the duration of treatment willdepend in part on the seriousness of the infection, the weight of thepatient, the duration of exposure of the recipient to the infectiousbacteria, and a variety of a number of other variables. The compositionmay be administered anywhere from once to several times a day, and maybe administered for a short or long term period. The usage may last fordays or weeks. Any dosage form employed should provide for a minimumnumber of units for a minimum amount of time. The concentration of theactive units or milligrams or micrograms of enzymes believed to providefor an effective amount or dosage of enzymes may be selected asappropriate. The amount of active units per mL (or micrograms/kilogrambody weight) and the duration of time of exposure depend on the natureof infection, and other factors that will be evident to those skilled inthe art given the benefit of this disclosure. In particular, dosageforms employed should provide for a minimum number of units for aminimum amount of time. The concentration of the active units of enzymebelieved to provide for an effective amount or dosage of enzyme may bein the range of any of the aforementioned weight/kg of body weight,and/or may comprise a suitable amount of enzymatic activity units/unitof volume.

The present invention encompasses antibacterial methods, includingmethods for killing of Gram-positive bacteria, for reducing a populationof Gram-positive bacteria, for treating or alleviating a bacterialinfection, for treating a human subject exposed to pathogenic bacteria,and for treating a human subject at risk for such exposure. In certainembodiments, a composition of this disclosure is administered to anindividual in need thereof. The individual can be a human individual forexample, who is diagnosed with, suspected of having, or is at risk forcontracting a bacterial infection. In other embodiments, the inventionincludes such approaches for use with non-human animals, including butnot limited to avian animals and mammals. In embodiments, the individualis a domesticated bird kept by humans for their eggs, their meat ortheir feathers (e.g., poultry). These birds are typically members of thesuperorder Galloanserae (fowl). Birds that can be treated usingcompositions of the invention thus include but are not limited tochickens, turkeys and ducks. In other embodiments, the non-human mammalis a companion animal, such as a feline or canine, guinea pigs, mice, orrabbits, or is livestock, including but not limited to bovine livestock.In embodiments, the disclosure includes administering separatecompositions comprising a lytic enzyme of this disclosure to apopulation of animals. In embodiments, the population of animalcomprises a population of bacteria that are in physical association witha polypeptide of this disclosure.

In embodiments, the disclosure pertains to prophylaxis and/or therapy ofan infection by any of C. perfringens, C. sordellii, or C. histolyticumthat is correlated with or is causative of any of food poisoning,gastrointestinal infections not associated with food, necroticenteritis, bacteremia, sepsis, toxic shock syndrome, gas gangrene,massive edema, pneumonia, endocarditis, arthritis, peritonitis,myonecrosis, or a gynecologic infection, or an infection of theumbilical stump in newborns.

Methods of using the therapeutic composition comprising a lyticenzyme/polypeptide(s) include administration by any acceptableapproaches including but not limited to topically, orally andparenterally. For example, the polypeptide(s) can be administeredintramuscularly, intrathecally, subdermally, subcutaneously,intravenously, or by aerosol to treat infections by Gram-positivebacteria. In embodiments, the disclosure comprises direct application ofthe polypeptide(s) using any suitable approaches to directly bring thepolypeptide in contact with the site of infection or bacterialcolonization, such as to the gastrointestinal tract, mucosa, applicationto a wound, application to the vaginal canal, expression by probiotics,and such.

Compositions for treating infections or contaminations comprise aneffective amount of at least one lytic enzyme according to theinvention, and may further comprise a carrier for delivering at leastone lytic enzyme to the infected or contaminated skin, coat, or internalgastrointestinal surface of an individual.

For compositions requiring absorption in the stomach and upper smallintestine and/or topical delivery to these sites, particularlycompositions with narrow absorption windows, bioadhesive, and/orgastroretentive drug delivery systems can be effective. Compositionsrequiring absorption or topical delivery only in the small intestine,enteric-coated, bioadhesive drug delivery systems can be utilized. Forcompositions requiring absorption or topical delivery only in the lowersmall intestine and colon enteric-coated, bioadhesive drug deliverysystems can be utilized. Pharmaceutical compositions of the inventionmay be, but are not limited to solutions, dispersions, emulsions,powders, pellets, beads, granules, tablets, compacts, sustained releaseformulations, capsules, microcapsules, tablets in capsules, tablets intablets, microspheres, shear form particles, floss, and flakes ormixtures thereof. Tablets include single layered tablets, multilayeredtablets, mini tablets, bioadhesive tablets, caplets, matrix tablets,tablet within a tablet, mucoadhesive tablets. Sustained releaseformulations include but are not limited to matrix type controlledrelease, membrane diffusion controlled release, site targeted,osmotically controlled release, pH dependent delayed release, timedrelease, pulsatile release, hydrodynamic balanced system; powders,pellets, beads, granules for suspension.

Compositions comprising polypeptides of this disclosure can be directedto the mucosal lining, where, in residence, they kill colonizing diseasebacteria. The mucosal lining includes, for example, the upper and lowerrespiratory tract, eye, buccal cavity, nose, rectum, vagina, periodontalpocket, intestines and colon. For birds, the compositions can beformulated for use on the skin and/or feather follicles.

Due to natural eliminating or cleansing mechanisms of mucosal tissues,conventional dosage forms may not be retained at the application sitefor any significant length of time. It may thus be advantageous to havematerials, which exhibit adhesion to mucosal tissues, to be administeredwith one or more polypeptides and other complementary agents over aperiod of time. The disclosure therefore includes use of mucoadhesives,including but not necessarily limited sustained release mucoadhesiveand/or bioadhesive formulations, which are known in the art.

Infections may also be treated by injecting into the infected tissue atherapeutic agent comprising the appropriate lyticenzyme(s)/polypeptide(s) and a carrier for the enzyme. The carrier maybe comprised of distilled water, a saline solution, albumin, a serum, orany combinations thereof. More specifically, solutions for infusion orinjection may be prepared in a conventional manner, e.g. with theaddition of preservatives such as p-hydroxybenzoates or stabilizers suchas alkali metal salts of ethylene-diaminetetraacetic acid, which maythen be transferred into fusion vessels, injection vials or ampules.Alternatively, the compound for injection may be lyophilized either withor without the other ingredients and be solubilized in a bufferedsolution or distilled water, as appropriate, at the time of use.Non-aqueous vehicles such as fixed oils, liposomes, and ethyl oleate arealso useful herein. Other phage associated lytic enzymes, along with aholin protein, may be included in the composition.

In an embodiment the disclosure comprises an article of manufacture thatcomprises at least one container, the container having within it apolypeptide of this disclosure which is optionally present in apharmaceutical formulation, the container optionally included printedmaterial having an indication that the polypeptide and/or thepharmaceutical formulation comprising it is for treating and/orpreventing an infection by Gram-positive bacteria, which may beindicated to be Clostridium perfringens, and which may further includean indication that the composition is used for veterinary purposes, suchas for treating a bacterial infection in avian animals. In embodiments,the avian animals are Anseriformes or Galliformes, and include but arenot limited to any type of waterfowl. In embodiments, the avian animalsare Galliformes and thus include any members of the order ofheavy-bodied ground-feeding birds that includes turkey, grouse, chicken,New World quail and Old World quail, ptarmigan, partridge, pheasant,junglefowl and the Cracidae. In embodiments, the avian animals aredomesticated fowl, including but not limited to domesticated chickensand turkeys. In embodiments, the chickens are roosters or hens. Inembodiments, the chickens are Gallus gallus, such as Gallus gallusdomesticus. In embodiments, the avian animals are adults, or juveniles.In embodiments, the avian animals are broiler chickens. In embodiments,a composition of this disclosure is administered to a population ofavian animals, i.e., a flock. In embodiments, from 50-100% members ofthe flock receive the composition.

In embodiments, compositions of this disclosure can be used fordetecting bacteria. In embodiments, the whole PlyCP025, or a segment ofit can be used for diagnostic approaches. In embodiments, the segmentcomprises the binding domain or the catalytic domain of PlyCP025.Variations in the amino acid sequences as described above are includedin connection with diagnostic approaches. In certain approaches suchpolypeptides are reversibly or irreversibly attached to a substrate. Thepolypeptides that have been reversibly or irreversibly attached to asubstrate may be in physical association with any bacteria as furtherdescribed herein. The substrate may be a component of a diagnosticdevice. Compositions comprising antibodies bound to polypeptides arealso included within the scope of this disclosure. In certain approachesthe PlyCP025 polypeptides described herein are components in animmunological assay, such as for use as a capture or detection agent in,for example, an ELISA assay. In certain approaches the PlyCP025polypeptides are detectably labeled. Any detectable label can be used,non-limiting examples of which include fluorescent labels, labels thatcan be detected via colorimetric assays, and polypeptides that canproduce a detectable signal, such as Green Fluorescent Protein, or anyother protein that produces a detectable signal. In embodiments,PlyCP025 polypeptides described herein are used in various diagnosticapproaches to determine the presence, absence, type and/or amount ofbacteria, including but not necessarily limited to any one or anycombination of C. perfringens, C. sordelli and C. histolyticum. Suchapproaches comprise contacting a sample with one or more PlyCP025polypeptides described herein such that a complex between a PlyCP025polypeptide and targeted bacteria (if present) is formed, and detectingthe PlyCP025 polypeptide that is bound to the bacteria. Any biologicalsample can be used. Suitable samples include but are not necessarilylimited to tissues and biological fluids. In embodiments, the samplecomprises blood, urine, saliva, lacrimal secretions, mucosa, esophagealfluid, feathers, skin or skin cells, or any combination thereof. Thesample can be obtained using any suitable technique and implement, suchas a needle or a swab. The sample can be used directly or can besubjected to a processing step prior to being analyzed. In embodiments,determining a value for binding of one or more PlyCP025 polypeptides tobacteria in a sample can be performed, and if desired can be compared toany suitable control to determine the presence, absence, amount and/ortype of bacteria in the sample.

The invention may be better understood by reference to the followingnon-limiting Examples, which are provided as exemplary of the invention.The following examples are presented in order to more fully illustratethe preferred embodiments of the invention and should in no way beconstrued, however, as limiting the broad scope of the invention.

Example 1

This Example describes the materials and methods used to produce theresults, which are described in the subsequent examples.

Bacterial Strains and Growth Conditions

Clostridium perfringens strains ATCC 13124 (type A), ATCC 3626 (type B),ATCC 27324 (type B), ATCC 3631 (type C), ATCC 51880 (type D),Clostridium difficile ATCC 43255 and Clostridium histolyticum ATCC 8034were obtained from ATCC. Two recent clinical isolates of Clostridiumperfringens were also used. Clostridium histolyticum, Clostridiumsepticum (ATCC 12464), Clostridium bifermentans (ATCC 638), andClostridium sordellii (ATCC 9714) were purchased from Microbiologics.Streptococcus pyogenes D471, Pseudomonas aeruginosa RS1, Bacillussubtilis SL4, Bacillus anthracis 1659 and ΔSterne, Bacillusthuringiensis 766, and Staphylococcus aureus RN4220 belong to theRockefeller University collection. The methicillin-susceptible S. aureus(MRSA) was also used. All strains were stored at −80° C. and cultivatedat 37° C. Staphylococcus, Streptococcus, Pseudomonas, and Bacillusstrains were cultivated in Difco brain heart infusion (BHI) broth(Spectrum). Escherichia coli was grown in Luria-Bertani (LB) broth (BDBiosciences). Clostridium strains were cultured in BHIS medium (BHIsupplemented with yeast extract [0.5%, wt/vol] and L-cysteine [10%,wt/vol]) and incubated in a Whitley A35 anaerobic chamber (MicrobiologyInternational, MD) supplied with an anaerobic gas mixture (10% CO2, 85%N2, 5% H2) (T.W. Smith).

Subcloning of the C. perfringens PlyCP025cat

The nucleotide sequence of PlyCP025 gene was acquired from the NCBIdatabase (WP_025647622.1). The protein sequence it encodes is givenabove as SEQ ID NO:1. The nucleotide sequence was synthesized, andinserted into a pUC57 vector after codon optimization for E. coliexpression (GenScript, NJ). After product verification by sequencing,the catalytic domain of PlyCP025, namely, PlyCP025-cat (SEQ ID NO:2given above), was generated by inserting a stop codon at the end of theamino acid sequence of the catalytic domain, Gly175, using a primer set5′-GCCGCCGAATTCATTATGAGCAAAATCTTTGGTCTGGATGC (SEQ ID NO:3) and3′-CGCCTGCAGTTAGCCGATGGTTTTGTCCAGCAGACCTTCGG (SEQ ID NO:4, listed in the5′ to 3′ direction). The construct was transformed into NEB 5-α F′Iqcompetent E. coli using EcoRI and PstI restrictive enzymes (New EnglandBioLabs). Positive clones were identified by colony PCR and sent for DNAsequencing (Genewiz, NJ).

The Expression and Purification of Recombinant PlyCP025 and itsCatalytic Domain

After nucleotide sequence verification, the aforementioned clones werepropagated in LB broth containing 100 μg/ml ampicillin to mid-log phase.The culture was then induced with 0.2% arabinose at 30° C. overnight.Cells were then pelleted, resuspended in 20 mM phosphate buffer (pH 7.0)containing EDTA-free complete Mini protease inhibitor cocktail (Roche),and lysed with an EmulsiFlex C-5 homogenizer (Avestin, Ottawa, Canada).Lysate debris was removed via ultracentrifugation at 4° C. (32,000×g, 45min), and the supernatant was sterile filtered through a 0.2-μm filter.The target protein was then purified from the supernatant with a HiTrapcation-exchange column (GE Healthcare, Uppsala, Sweden). Lysin waseluted using a stepwise gradient of 0.0 to 1.0 M NaCl in 50 mM phosphatebuffer (PB) (pH 7.0). Extra salt was removed by filtration with a10-kDa-cutoff Ultra Centricon (Amicon).

Lytic Activity Assays

C. perfringens ATCC13124 strain was grown to mid-log phase underanaerobic conditions, and cells were harvested by centrifugation(3,000×g, 5 min). Pellets were washed twice and resuspended in PB togenerate a final optical density at 600 nm (OD600) of approximately 0.9.The lytic activity of lysin was calculated based on reduction in OD600as measured in 96-well plates using a SpectraMax Plus reader (MolecularDevices, Sunnyvale, Calif.). For each sample, 20 μl of lysin (50 μg/mlfinal concentration) or an equal volume of 20 mM PB was added to 180 μlof cell resuspension. The drop in OD600 at 37° C. was measured once perminute for 60 min.

To study the effect of pH on the activity of lysin, the sameexperimental conditions and optical drop assays described above wereperformed with buffers of different pH, i.e., 20 mM PB (pH 6.0, 7.0, and8.0). To study the effect of salt and cation concentrations on theactivity of lysin, PB (pH 8.0) that contained various concentrations ofNaCl (50 mM, 100 mM, 200 mM or 400 mM) was used. All experiments wereperformed in duplicates.

CFU Reduction Assay

C. perfringens ATCC13124 strain was grown to mid-log phase underanaerobic conditions, and cells were harvested by centrifugation(3,000×g, 5 min). The pellet was washed twice and resuspended in PB to afinal concentration of 10⁸ cfu/ml. For each sample, 20 μl of lysin (50μg/ml final concentration) or an equal volume of 20 mM PB was added to180 μl of cell resuspension for lytic reaction. Every 15 minutes, 50 μlwas sampled from the reaction and performed serial dilution in 20 mM PB.25 μl from each serial dilution was then plated onto BHI agar plate.After overnight incubation in the anaerobic condition, the number ofcolony at each time point was counted from the dilution that contains20-40 colonies. The final bacterial titer was calculated based on asummation of the dilution fold and the colony count.

Example 2

This Example describes the expression and purification of PlyCP025catfrom E. coli. The DNA sequences of PlyCP025cat was cloned into a pBAD24expression vector, and after arabinose induction, the whole-cell lysatewas individually purified by cation-exchange chromatography. Elutedfractions that contained the purified target proteins (PlyCP025cat [19kDa]) were pooled and run on SDS-PAGE. The final purified productswere >90% pure (FIG. 1). The average yield was about 20 mg protein perliter of E. coli culture. These purified molecules were used in allsubsequent experiments. A comparison of the activity of PlyCP025 andPlyCP025cat is shown in FIG. 4.

Example 3

This Example provides a molecular characterization of PlyCP025cat.

Lytic Activity in CFU Reduction

To measure the lytic activity of PlyCP025cat directly, a CFU reductionassay was performed. PlyCP025cat reduced the CFU of C. perfringens by 1log every 15 minutes approximately (FIG. 1). In 60 min, 50 μg/mlPlyCP025cat significantly reduced the titer of C. perfringens, from 10⁸to 10⁴, suggesting strong lytic activity against live C. perfringens.

Effects of pH and Salt

To investigate the lytic activity of PlyCP025cat under variousconditions and identify the optimal activity condition, the reduction inthe OD600 of C. perfringens suspensions was monitored over 60 min.PlyCP025cat displayed the strongest activity at pH 8.0, followed bypH7.0, and showed moderate activity at pH 6.0 (FIG. 2A).

To test salt sensitivity, the lytic activity of PlyCP025cat was measuredwith different NaCl concentrations. While PlyCP025cat displayedequivalent lytic activity at the end of 60 min for all concentrationstested (0 to 200 mM NaCl), greater lytic activity was observed duringthe initial 10 min of the reaction with minimal NaCl (FIG. 2B). At 400mM and 500 mM NaCl, the lytic activity of PlyCP025cat was inhibitedsignificantly.

Example 4

This Example demonstrates host specificity. To study the hostspecificity of PlyCP025cat, we tested its lytic activity againstmultiple C. perfringens strains, both laboratory and clinical strains,other Clostridium spp and several common gut flora species. Allclostridium species were cultured anaerobically until mid-exponentialphase, washed, and resuspended in 20 mM PB, pH 7.0. PlyCP025cat was thenadded to the bacterial suspensions at a final concentration of 50 μg/ml,and the OD600 values of each culture were recorded over 60 min. Theratio of OD600 at 60 min versus time zero was calculated. PlyCP025catdemonstrated the effective lytic activity against the majority of C.perfringens strains, as well as C sordelli and C. histolyticum, butdisplayed little to no activity against C. septicum, C bifermentans andC. difficile (FIG. 3). PlyCP025cat displayed no activity against othercommon gut flora species, including B. subtillis, B anthracis ΔSterne,P. aeruginosa, MRSA, Staphylococcus aureus RN4220, S pyogen D471, Sepidermidis HER 1292 and B. thuringiensis 766. As an exception,PlyCp025cat displayed mild lytic activity against B. anthracis 1659strain.

Although the present disclosure has been described with respect to oneor more particular embodiments and/or examples, it will be understoodthat other embodiments and/or examples of the present disclosure may bemade without departing from the scope of the present disclosure.

1. A method for reducing one or more of C. perfringens, C. sordelli orC. histolyticum bacteria, the method comprising contacting the bacteriawith a composition comprising a lytic enzyme that comprises the aminoacid sequence of SEQ ID NO: 2 or an amino acid sequence with acontiguous segment having at least 95% identity to the amino acidsequence of SEQ ID NO:2 such that one or more of the one or more of theC. perfringens, C. sordelli or C. histolyticum bacteria are killed. 2.The method of claim 1, wherein the lytic enzyme consists of the sequenceof SEQ ID NO:2 or an amino acid sequence with a contiguous segmenthaving at least 95% identity to the amino acid sequence of SEQ ID NO:2.3. The method of claim 1 wherein at least the C. perfringens bacteriaare killed.
 4. The method of claim 3, wherein the bacteria areantibiotic resistant.
 5. The method claim 4, wherein at least the C.perfringens bacteria are antibiotic-resistant and are killed.
 6. Themethod of claim 1, wherein the bacteria are killed are in or on a human,or in or on a non-human animal selected from avian animals and non-humanmammals.
 7. The method of claim 6, wherein the bacteria are in theindividual in a population of bacteria that further comprise commensalgut bacterium and wherein the commensal gut bacteria are not killed bythe lytic enzyme.
 8. The method of claim 7, wherein the commensalbacteria are selected from C. septicum, C. novyi, E. faecalis, E.faecium, L. rhamnosous, and combinations thereof.
 9. A pharmaceuticalcomposition for killing C. perfringens, C. sordelli or C. histolyticumor a combination thereof comprising a lytic enzyme that comprises theamino acid sequence of SEQ ID NO: 2 or an amino acid sequence with atleast 95% identity to the amino acid sequence of SEQ ID NO:2, thecomposition further comprising at least one pharmaceutically acceptablecarrier or excipient.
 10. The pharmaceutical composition of claim 9,wherein the lytic enzyme consists of the sequence of SEQ ID NO:2 or asequence with at least 95% identity to the amino acid sequence of SEQ IDNO:2.
 11. The pharmaceutical composition of claim 9, wherein the lyticenzyme is produced by an expression vector.
 12. A method of making arecombinant polypeptide capable of killing C. perfringens, C. sordelliand C. histolyticum comprising expressing the recombinant polypeptide ina population of cells comprising an expression vector that encodes andexpresses the recombinant polypeptide, wherein the recombinantpolypeptide comprises an amino acid sequence of SEQ ID NO: 2 or an aminoacid sequence with at least 95% identity to the amino acid sequence ofSEQ ID NO:2, and separating the recombinant polypeptide from thepopulation of cells.
 13. The method of claim 12, wherein the recombinantpolypeptide consists of SEQ ID NO:2 or an amino acid sequence with atleast 95% identity to the amino acid sequence of SEQ ID NO:2.
 14. Anexpression vector encoding a polypeptide of claim
 12. 15. A bacteriumcomprising the expression vector of claim
 14. 16. A single bacterium, ora population of bacteria, in physical association with a polypeptidethat comprises the amino acid sequence of SEQ ID NO: 2 or an amino acidsequence with a segment having at least 95% identity to the amino acidsequence of SEQ ID NO:2.
 17. The single bacterium, or the population ofbacteria of claim 16, wherein the polypeptide consists of the amino acidsequence of SEQ ID NO: 2 or an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO:2.
 18. The populationof bacteria of claim 17, wherein the population of bacteria are presenton an avian animal.
 19. The population of bacteria of claim 18, whereinthe polypeptide consists of the amino acid sequence of SEQ ID NO:
 2. 20.A composition comprising lysed bacteria and a polypeptide consisting ofthe sequence of SEQ ID NO:1 or SEQ ID NO:2.
 21. A method comprisingcontacting a sample with a polypeptide comprising the amino acidsequence of SEQ ID NO:1 or SEQ ID NO: 2 or an amino acid sequence with acontiguous segment having at least 95% similarity to SEQ ID NO:1 or SEQID NO: 2, and detecting binding of the polypeptide to bacteria in thesample if said bacteria that are bound to the polypeptide are present inthe sample.
 22. The method of claim 21, wherein the polypeptide isdetectably labeled, and wherein a signal from the detectable label isdetected if bacteria that are bound to the polypeptide are present inthe sample.